The protein product of c-Met oncogene is the tyrosine kinase receptor for hepatocyte growth factor (HGF) also known as scatter factor (SF). HGF and its receptor c-Met are widely expressed in a variety of tissues, and their expression is normally confined to cells of mesenchymal and epithelial origin, respectively. The HGF-Met pathway is involved in a wide range of biological effects, including cell proliferation and survival, cell adhesion, cell migration and invasion, morphogenic differentiation, organization of tubular structures and angiogenesis. Such paracrine signaling is vital to normal embryogenic development, wound healing and tissue maintenance and regeneration (reviewed in Christensen et al, 2005, Cancer Letters 225: 1-26).
While HGF-Met signaling plays a key role during normal development, inappropriate activation of this signaling pathway has been implicated in tumor development and progression. Aberrant c-Met signaling has been described in a variety of human cancers, including solid tumors and hematologic malignancies. Met activation may be involved in different stages of tumor progression, such as tumor cell proliferation and survival in primary tumors, induction of angiogenesis, stimulation of cell motility to form micrometastases, induction of invasive phenotype, and regaining the proliferation phenotype to form overt metastases (Birchmeier et al 2003, Nat. Rev. Mol. Cell. Biol. 4: 915-925).
Several mechanisms cause dysregulation of the HGF-Met pathway in tumor cells, such as overexpression of c-Met and/or HGF, constitutive kinase activation of c-Met in the presence or absence of gene amplification, activating mutations of c-Met, and autocrine activation of c-Met by HGF. c-Met is expressed in most carcinomas, but the degree of expression varies among distinct tumor types. High expression is detected in renal and colorectal carcinomas, and lung adenocarcinomas. Overexpression of ligand and/or receptor correlates with high tumor grade and poor prognosis. c-Met mutations have been reported in several types of tumors, such as hereditary and sporadic human papillary renal carcinomas, as well as ovarian cancer, childhood hepatocellular carcinoma, head and neck squamous cell carcinomas, gastric and lung cancers (reviewed in Maulik et al, 2002. Cytokine & Growth Factor Rev. 13: 41-59; Ma et al, 2003. Cancer and Metastasis Rev. 22: 309-325).
The HGF-Met pathway is involved in cell scattering. HGF was discovered as a secretory product of fibroblasts and smooth muscle cells that induces dissociation and motility of epithelial cells. It is able to induce cell dissociation and mutual repulsion in a similar manner to semaphorins. HGF-Met signaling is also involved in cell motility. The key events regulating cell motility are polymerization of actin, formation of actin stress fibers, and focal adhesion formation. HGF has been shown to induce branching morphogenesis of kidney, mammary and bile ductular cells. In response to HGF, Met-expressing cells form branches in three-dimensional matrigel or tubule-like structures in collagen gels. This process is mediated through changes in cell shape, asymmetric polarization of the cells in the direction of branching, branch elongation, cell-cell contact, cell-ECM communication, ECM remodeling, controlled proteolysis and cell motility (Zhang et al. 2003. J. Cell. Biochem., 88:408-417; Ma et al, 2003. ibid). HGF acts as a potent angiogenic factor. HGF stimulation of vascular endothelial cells promotes migration, proliferation, protease production, invasion, and organization into capillary-like tubes. HGF can also promote the expression of angiogenic factors by tumor cells (Ma et al, 2003. ibid).
HGF-Met signaling has been strongly implicated in the promotion of the invasive/metastatic tumor phenotype. An HGF-stimulated pathway involving MAPK1/2 signaling is important in the up-regulation of expression of the serine protease urokinase (uPA) and its receptor (uPAR), resulting in an increase of uPA on the cell surface. Certain components of the ECM can be directly degraded by uPA, and more importantly, uPA cleaves plasminogen into the broader-specificity protease plasmin, which is able to efficiently degrade several ECM and basement membrane (BM) components. Plasmin also activates metalloproteinases, which have potent ECM/BM degrading abilities. HGF has been reported to promote attachment of tumor cells to endothelium, an important step in the metastatic cascade. This activity may be mediated by HGF induced up-regulation of CD44 expression on endothelium cells, and integrin expression on tumor cells.
The human Met gene, which includes 21 exons, is located on chromosome 7 band 7q21-q31 and spans more than 120 kb in length. The primary Met transcript produces a 150 kDa polypeptide (1390 amino acids) that is partially glycosylated to produce a 170 kDa precursor protein. This 170 kDa precursor is further glycosylated and then cleaved into a 50 kDa α-chain and a 140 kDa β-chain which are disulfide-linked. The α-subunit of the mature Met heterodimer is highly glycosylated and is entirely extracellular, while the β-subunit contains a large extracellular region, a membrane spanning segment, and an intracellular tyrosine kinase domain (Ma et al, 2003. ibid).
Met is the prototypic member of a subfamily of heterodimeric receptor tyrosine kinases which include Met, Ron, and Sea. Members of the Met receptor subfamily have been shown to share homology with semaphorins and semaphorin receptors (plexin), which play a role in cell scattering (Reviewed in Trusolino et al. 1998, FASEB J. 12: 1267-1280). All semaphorins contain a conserved 500 amino acid extracellular domain (Sema domain), which spans the cysteine-rich Met related sequence (MRS), containing the consensus motif C—X(5-6)-C—X(2)-C—X(6-8)-C—X(2)-C—X(3-5)-C. The extracellular portions of Met, Ron, and Sea contain a region of homology to semaphorins including the N-terminal Sema domain and the MRS. Other domains identified in the extracellular portion of Met are the PSI domain and the IPT/TIG repeat domain. The PSI domain is found in plexins, semaphorins and integrins while the IPT repeats (also known as TIG domains) are found within immunoglobulin, plexins and transcription factors. The C-terminus intracellular tyrosine kinase domain shares homology with Ron and Sea.
The Sema domain plays a critical role in ligand binding and is also necessary for receptor dimerization (Kong-Beltran et al 2004, Cancer Cell, 6: 75-84; Wickramasinghe and Kong-Beltran, 2005, Cell Cycle, 4: 683-685). Treatment of Met-overexpressing tumor cells with a recombinant Sema protein construct (rSema, which contains also the PSI domain) inhibits both ligand dependent and independent activation of Met-mediated signal transduction, cell motility and migration, in a manner similar to the antagonistic anti-Met Fab 5D5 (Kong-Beltran et al 2004. ibid). Decoy Met (the entire extracellular domain of Met, produced as a truncated soluble receptor) interferes with HGF binding to Met, and with receptor dimerization. Similarly, a chimeric soluble protein containing the extracellular domain of Met fused to the constant region of IgG heavy chain, binds HGF with an affinity similar to that of the authentic, membrane-associated receptor, and inhibits the binding of HGF to Met, expressed on A549 cells (Mark, et al., 1992, J Biol. Chem. 267:26166-26171). Local or systemic delivery of decoy Met in mice, by lentiviral vector technology, inhibits tumor cell proliferation and survival in a variety of human xenografts, impairs tumor angiogenesis, suppresses or prevents the formation of spontaneous metastases, and synergizes with radiotherapy in inducing tumor regression (Michieli et al, 2004, Cancer Cell 6: 61-73). These data suggest that the extracellular domain of Met may not only represent a novel anticancer therapeutic target, but also acts as a biotherapeutic itself (reviewed in Zhang et al 2004, Cancer Cell 6: 5-6).
Various inhibitory strategies have been employed to therapeutically target the HGF-Met pathway (reviewed in Christensen et al, 2005, Cancer Letters 225: 1-26), and several candidates are under development. Three main approaches have been employed for selective anticancer drug development: antagonism of HGF/Met interaction, inhibition of tyrosine kinase catalytic activity of Met, and blockade of intracellular Met/effectors interactions. Among the current developments are a humanized anti-HGF mAb AMG-102 (Amgen); NK4, a proteolytic cleavage fragment of HGF that acts as a competitive HGF antagonist (Kringle Pharma); and small molecule inhibitors of the c-Met receptor, such as XL880 (Exelixis), ARQ 197 (Arqule), SU11274, PHA665752, PF-02341066 of Pfizer; a series of small molecules of Methylgene, and others.
WO 2005/113596 assigned to Receptor Biologix Inc, discloses several in silico predicted polypeptides that are isoforms of cell surface receptors, including, inter alia, Met receptor, wherein each polypeptide comprises at least one domain of the receptor, operatively linked to at least one amino acid encoded by an intron of a relevant gene; and the polypeptide lacks a transmembrane domain, protein kinase domain and at least one additional domain compared to the wt receptor, whereby the membrane localization and protein kinase activity of the polypeptide is reduced or abolished compared to the receptor. It is further speculated that these isoforms may be useful in treating or preventing metastatic cancer, inhibiting angiogenesis, treating lung cancer, malignant peripheral nerve sheath tumors, colon cancer, gastric cancer, cutaneous malignant melanoma and prevention of malaria. WO 2005/113596 mentions that the Met isoforms might be provided in pharmaceutical compositions as conjugates between the isoform and another agent, including coupling to an Fc fragment of an antibody that binds to a specific cell surface marker to induce killer T cell activity in neturophils, natural killer cells and macrophages. However, no guidance is provided for production of any conjugates, nor are there any examples for actual biological activities of said Met isoforms.
U.S. Pat. No. 5,571,509 assigned to Farmitalia Carlo Erba S. R. L., discloses a carboxy-terminal truncated form of the c-Met oncogene. The truncated form results in a beta chain of the receptor, which is 75 to 85 kDa long that acts as an antagonist of the HGF receptor. U.S. Pat. No. 5,571,509 reveals that this soluble Met protein is released in the culture medium by proteolytic cleavage of the membrane-bound Met proteins. However, these proteolytic fragments are not novel splice variants of cMet.
US Patent Application Publication No. 2005/0233960 assigned to GENETECH, INC. discloses c-Met antagonists for modulating the HGF/c-met signaling pathway. The c-Met antagonists of US 2005/0233960 are particularly peptides comprising at least a portion of c-Met Sema domain or variant thereof.
There is an unmet need to develop therapies which target the HGF-Met pathway and Met signaling via Met receptor tyrosine kinase, and which inhibit Met receptor action and/or its physiological effects.